In 2011, we continued to delete additional secreted proteases from B. anthracis. Strains lacking multiple proteases were used to define the contributions of individual proteases to the degradation of proteins in the secretome. In this study we showed that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the attenuated B. anthracis Ames 35 variant strain, are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). This allowed the role of each protease in the degradation of the B. anthracis toxin components and ALO to be determined. In the final strain constructed, which lacks six proteases, the levels of the anthrax toxin components and ALO in the supernatant were significantly increased and remained stable over 24 h. For general use as a host for the production of recombinant proteins, the strain was cured of pXO1 and made permanently sporulation deficient. This strain, designated BH460, has proven useful in the preparation of a number of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or E. coli hosts. BH460 is therefore recommended as an effective host strain for recombinant protein production, typically yielding greater than 10 mg pure protein per liter of culture. Work is underway to obtain intellectual property protection and to license this strain to several commercial entities. In a separate project, we continued analyses of the genes and sequences that are needed for replication and maintenance of the key virulence plasmid pXO1, which encodes all three anthrax toxin proteins. In previous work we identified and isolated the region of pXO1 that is responsible for plasmid replication, and thereby defined a minireplicon that was different from one reported by others. It has now become clear through the work of others that the minireplicon we defined is universally conserved among the large family of similar bacillus species plasmids, validating our identification. However, DNA replication alone does not assure that every daughter cell receives a copy of pXO1 when cell division occurs. Thus, we seek to understand the mechanism that causes accurate segregation of plasmids between daughter cells, an active process that assures plasmid maintenance in the bacterial population. Decreased segregational stability of the pXO1 minireplicon in comparison with the native pXO1 was observed in B. anthracis during repeated passage at 37C. This is likely to result from the absence of a functional plasmid maintenance system within the minireplicon region. During the current reporting period of 2011, we adopted the Saccharomyces cerevisiae Flp-FRT recombination system to the genetic manipulation of B. anthracis. Using this system along with the Cre-loxP system, two distinct areas responsible for pXO1 maintenance were identified on the plasmid. The exact locations of these new genetic elements are being determined by constructing and testing additional deletions. This knowledge will provide targets for anti-infective agents - those that do not directly kill the pathogen but instead render it less virulent, thereby allowing host immune responses to effectively combat it.